The present invention pertains to improved methods for detecting nucleic acid sequences in biological samples, particularly sequences derived from infectious microorganisms.
Human Immunodeficiency Virus (HIV) infects millions of individuals world-wide and consequently represents a serious public health concern. Spread of HIV infection via contaminated blood products means that there is a need for screening methods that can detect small amounts of HIV RNA in patient samples. Furthermore, the increasing availability of ameliorative treatments for HIV infection means that early detection of infection in a patient is vital in order to initiate appropriate therapeutic interventions.
Hepatitis C Virus (HCV) is a parenterally transmitted virus responsible for the majority of cases of post-transfusion hepatitis and a substantial portion of sporadic (or community acquired) hepatitis cases worldwide. It is estimated that more than 1% of the world""s population is infected with HCV. HCV infection is associated with acute hepatitis, chronic hepatitis, cirrhosis, and subsequent hepatocellular carcinoma.
HCV is currently classified as a separate genus, Hepacivirus, in the family Flaviviridae. Its genome consists of a positive-stranded RNA molecule of about 9,500 nucleotides with a single, large open reading frame (ORF) which encodes a polyprotein precursor of about 3,000 amino acids. The large ORF is preceded by a 5xe2x80x2 non-coding (NC) region of about 340 nucleotides, which is the most highly conserved region of the genome. The 5xe2x80x2 region of the ORF encodes (in a 5xe2x80x2-to-3xe2x80x2 direction) a capsid protein, two envelope glycoproteins (E1 and E2), and a small protein of unknown function (P7). The 3xe2x80x2 portion of the ORF encodes nonstructural proteins which include a protease, protease/helicase bi-functional protein, RNA polymerase, and regulatory peptides.
Analysis of HCV coding sequences from around the world has revealed considerable sequence variation among individual viral isolates. Furthermore, analyses of HCV sequences from individual patients have shown that the virus circulates as so-called xe2x80x9cquasi-species,xe2x80x9d which contain related but not identical sequences. The variation that exists among isolates and within individual patients is believed to be the result of the low fidelity of the virally-encoded RNA-dependent RNA polymerase. The degree of genetic variability of HCV has important implications for prevention, diagnosis, and control of infection.
Serodiagnosis of HCV infection is typically determined by commercially available enzyme immuno-assays (EIA) which detect antibodies that bind recombinant HCV proteins or peptides. Positive EIA results can be confirmed by a recombinant immunoblot assay (RIBA), but neither EIA nor RIBA assays distinguish past from present infections. Because of the typically low titer of circulating virus, a direct assay for viral proteins has not been successfully developed. Furthermore, antibody-based assays usually fail to detect HCV infection for 2 to 3 months after exposure.
Thus, there is a need in the art for improved assays that allow for the simultaneous screening of patient samples for both HIV and HCV.
The present invention provides methods for the simultaneous detection of the presence of Hepatitis C Virus (HCV) RNA and Human Immunodeficiency Virus (HIV) RNA in a biological sample using a multiplex assay.
Thus, in one aspect, the invention is directed to a method for co-detecting Hepatitis C Virus (HCV) RNA and Human Immunodeficiency Virus (HIV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using RNA derived from the sample as a template and at least one reverse transcription primer that will prime reverse transcription of DNA from HCV RNA and at least one reverse transcription primer that will prime reverse transcription of DNA from HIV RNA to produce reverse transcription products comprising (a) HCV-specific reverse transcription products, (b) HIV-specific reverse transcription products, or (c) a combination of (a) and (b);
(B) amplifying the reverse-transcription products using one or more pairs of oligonucleotide primers specific for the 5xe2x80x2 noncoding region of HCV and one or more pairs of oligonucleotide primers specific for HIV to produce amplification products comprising (a) HCV-specific amplification products, (b) HIV-specific amplification products, or (c) a combination of (a) and (b);
where each of the pairs of oligonucleotide primers specific for HCV comprises:
where each of the pairs of oligonucleotide primers specific for HIV-1 comprises a forward primer with the sequence:
5xe2x80x2-CTGCTTAAGCCTCAATAAAGCTTGCCTTGA-3xe2x80x2 (JBLTR4)  less than SEQ ID NO. 3 greater than , and a reverse primer specific for HIV-1 selected from the group consisting of
(1) 5xe2x80x2-GGGTCTGAGGGATCTCTAGTTACC AGAGT-3xe2x80x2 (JBLTR6)  less than SEQ ID NO. 4 greater than , and
(2) 5xe2x80x2-TGTTCGGGCGCCACTGCTAGAGA-3xe2x80x2 (JBLTR8)  less than SEQ ID NO. 5 greater than , and where each of the pairs of oligonucleotide primers specific for HIV-2 comprises a forward primer with the sequence 5xe2x80x2-GGGAGGTTCTCTCCAGCACTAGCA-3xe2x80x2 (2LTRe)  less than SEQ ID NO. 6 greater than , and a reverse primer specific for HIV-2 with the sequence:
5xe2x80x2-GCGACTAGGAGAGATGGGAACACACA-3xe2x80x2 (2LTR-R1)
 less than SEQ ID NO. 7 greater than ; and
(C) detecting the amplification products, where detection of HCV-specific amplification products indicates the presence of HCV RNA in the sample, detection of HIV-specific amplification products indicates the presence of HIV RNA in the sample, and the detection of HCV-specific amplification products and HIV-specific amplification products indicates the presence of HCV RNA and HIV RNA in the sample.
In a second aspect, the invention is directed to a method for co-amplifying Hepatitis C Virus (HCV) DNA and Human Immunodeficiency Virus (HIV) DNA. The method comprises:
(A) performing a polymerase chain reaction on a DNA sample suspected to contain HCV DNA, HIV DNA, or a combination of HCV DNA and HIV DNA, using one or more pairs of oligonucleotide primers specific for the 5xe2x80x2 noncoding region of HCV and one or more pairs of oligonucleotide primers specific for HIV to produce amplification products comprising (a) HCV-specific amplification products, (b) HIV-specific amplification products, or (c) a combination of (a) and (b);
where each of the pairs of oligonucleotide primers specific for HCV comprises:
(i) forward primer 5xe2x80x2-GGGAGAGCCATAGTGGTCTGCGGAA-3xe2x80x2 (C131F25)  less than SEQ ID NO. 1 greater than , and
(ii) reverse primer 5xe2x80x2-CGGGGCACTCGCAAGCACCCTATCA-3xe2x80x2 (C294R25)  less than SEQ ID NO. 2 greater than ;
where each of the pairs of oligonucleotide primers specific for HIV-1 comprises a forward primer with the sequence:
5xe2x80x2-CTGCTTAAGCCTCAATAAAGCTTGCCTTGA-3xe2x80x2 (JBLTR4)  less than SEQ ID NO. 3 greater than , and a reverse primer specific for HIV-1 selected from the group consisting of:
(1) 5xe2x80x2-GGGTCTGAGGGATCTCTAGTTACC AGAGT-3xe2x80x2 (JBLTR6)  less than SEQ ID NO. 4 greater than , and
(2) 5xe2x80x2-TGTTCGGGCGCCACTGCTAGAGA-3xe2x80x2 (JBLTR8)  less than SEQ ID NO. 5 greater than , and where each of the pairs of oligonucleotide primers specific for HIV-2 comprises a forward primer with the sequence 5xe2x80x2-GGGAGGTTCTCTCCAGCACTAGCA-3xe2x80x2 (2LTRe)  less than SEQ ID NO. 6 greater than , and a reverse primer specific for HIV-2 with the sequence:
5xe2x80x2-GCGACTAGGAGAGATGGGAACACACA-3xe2x80x2 (2LTR-R1)  less than SEQ ID NO. 7 greater than .
In a third aspect, the invention is directed to a method for co-detecting Hepatitis C Virus (HCV) RNA and Human Immunodeficiency Virus (HIV) RNA in a biological sample. The method comprises:
(A) performing a reverse transcription reaction using RNA derived from the sample and internal positive control (IPC) RNA as a template, at least one reverse transcription primer that will prime reverse transcription of DNA from IPC RNA, at least one reverse transcription primer that will prime reverse transcription of DNA from HCV RNA, and at least one reverse transcription primer that will prime reverse transcription of DNA from HIV RNA, to produce reverse transcription products comprising (a) IPC-specific reverse transcription products and (b) HCV-specific reverse transcription products, (c) HIV-specific reverse transcription products, or (d) a combination of any of the foregoing;
(B) amplifying the reverse-transcription products using one or more pairs of oligonucleotide primers specific for IPC, one or more pairs of oligonucleotide primers specific for the 5xe2x80x2 noncoding region of HCV, and one or more pairs of oligonucleotide primers specific for HIV to produce amplification products comprising (a) IPC-specific amplification products (b) IPC-specific amplification products and HCV-specific amplification products, (c) IPC-specific amplification products and HIV-specific amplification products, or (d) a combination of any of the foregoing;
where each of the pairs of oligonucleotide primers specific for IPC comprises:
(i) forward primer 5xe2x80x2-CGCCAGCGTGGACCATCAAGT AGTAA-3xe2x80x2 (IPCF1)  less than SEQ ID NO. 8 greater than , and
(ii) reverse primer 5xe2x80x2-CACGATCCTGGAGCAGACACT GAAGA-3xe2x80x2 (IPCR1)  less than SEQ ID NO. 9 greater than ;
where each of the pairs of oligonucleotide primers specific for HCV comprises:
(i) forward primer 5xe2x80x2-GGGAGAGCCATAGTGGTCT GCGGAA-3xe2x80x2(C131F25)  less than SEQ ID NO. 10 greater than , and
(ii) reverse primer 5xe2x80x2-CGGGGCACTCGCAAGCACC CTATCA-3xe2x80x2 (C294R25)  less than SEQ ID NO. 11 greater than ;
where each of the pairs of oligonucleotide primers specific for HIV-1 comprises a forward primer with the sequence:
5xe2x80x2-CTGCTTAAGCCTCAATAAAGCTTGCCTTGA-3xe2x80x2 (JBLTR4),  less than SEQ ID NO. 3 greater than  and a reverse primer specific for HIV-1 selected from the group consisting of:
(1) 5xe2x80x2-GGGTCTGAGGGATCTCTAGTTACC AGAGT-3xe2x80x2 (JBLTR6)  less than SEQ ID NO. 4 greater than , and
(2) 5xe2x80x2-TGTTCGGGCGCCACTGCTAGAGA-3xe2x80x2 (JBLTR8)  less than SEQ ID NO. 5 greater than ,and where each of the pairs of oligonucleotide primers specific for HIV-2 comprises a forward primer with the sequence 5xe2x80x2-GGGAGGTTCTCTCCAGCACTAGCA-3xe2x80x2 (2LTRe)  less than SEQ ID NO. 6 greater than , and a reverse primer specific for HIV-2 with the sequence:
5xe2x80x2-GCGACTAGGAGAGATGGGAACACACA-3xe2x80x2 (2LTR-R1)  less than SEQ ID NO. 7 greater than ; and
(C) detecting the amplification products, where detection of IPC-specific amplification products indicates the presence of IPC RNA in the sample, detection of HCV-specific amplification products indicates the presence of HCV RNA in the sample, detection of HIV-specific amplification products indicates the presence of HIV RNA in the sample, and the detection of HCV-specific amplification products and HIV-specific amplification products indicates the presence of HCV RNA and HIV RNA in the sample.
In a fourth aspect, the invention is directed to a method for co-amplifying Internal Positive Control (IPC) DNA, Hepatitis C Virus (HCV) DNA, and Human Immunodeficiency Virus (HIV) DNA. The method comprises:
(A) performing a polymerase chain reaction on a DNA sample suspected to contain IPC DNA, HCV DNA, HIV DNA, or a combination of any of the foregoing, using one or more pairs of oligonucleotide primers specific for IPC, one or more pairs of oligonucleotide primers specific for the 5xe2x80x2 noncoding region of HCV, and one or more pairs of oligonucleotide primers specific for HIV to produce amplification products comprising (a) IPC amplification products, (b) HCV-specific amplification products, (c) HIV-specific amplification products, or (d) a combination of any of (a), (b), and (c);
where each of the pairs of oligonucleotide primers specific for IPC comprises:
where each of the pairs of oligonucleotide primers specific for HCV comprises:
(i) forward primer 5xe2x80x2-GGGAGAGCCATAGTGGTCTGCGGAA-3xe2x80x2 (C131F25)  less than SEQ ID NO. 10 greater than , and
(ii) reverse primer 5xe2x80x2-CGGGGCACTCGCAAGCACCCTATCA-3xe2x80x2 (C294R25)  less than SEQ ID NO. 11 greater than ;
where each of the pairs of oligonucleotide primers specific for HIV-1 comprises a forward primer with the sequence:
5xe2x80x2-CTGCTTAAGCCTCAATAAAGCTTGCCTTGA-3xe2x80x2 (JBLTR4)  less than SEQ ID NO. 3 greater than , and a reverse primer specific for HIV-1 selected from the group consisting of:
(1) 5xe2x80x2-GGGTCTGAGGGATCTCTAGTTACC AGAGT-3xe2x80x2 (JBLTR6)  less than SEQ ID NO. 4 greater than , and
(2) 5xe2x80x2-TGTTCGGGCGCCACTGCTAGAGA-3xe2x80x2 (JBLTR8)  less than SEQ ID NO. 5 greater than , where each of the pairs of oligonucleotide primers specific for HIV-2 comprises a forward primer with the sequence 5xe2x80x2-GGGAGGTTCTCTCCAGCACTAGCA-3xe2x80x2 (2LTRe)  less than SEQ ID NO. 6 greater than , and a reverse primer specific for HIV-2 with the sequence:
5xe2x80x2-GCGACTAGGAGAGATGGGAACACACA-3xe2x80x2 (2LTR-R1)  less than SEQ ID NO. 7 greater than .
In other aspects of the invention, the reverse transcription reaction is performed using random oligonucleotide primers; alternatively, one or more HCV-specific and one or more HIV-specific reverse transcription primers, i.e., oligonucleotides having sequences that correspond to sequences in HCV or HIV RNA, may be used.
Methods for detection of amplification include, without limitation, (a) electrophoresis and (b) capture of amplification products on a solid support to which IPC-, HCV- or HIV-specific probes are attached followed by quantifying the bound products using a colorimetric assay. Useful IPC-specific capture probes include, without limitation, 5xe2x80x2-CTGCGTTAGACCGAGAACTGTGGATAAAGG-3xe2x80x2  less than SEQ ID NO. 17 greater than . Useful HCV-specific 5xe2x80x2 capture probes include, without limitation, 5xe2x80x2-CCTTTCGCGACCCAACACTACTCGGCT-3xe2x80x2  less than SEQ ID NO. 12 greater than . Useful HIV-1 specific capture probes include, without limitation, 5xe2x80x2-CAACAGACGGGCACACACTACT-3xe2x80x2  less than SEQ ID NO. 13 greater than  and useful HIV-2 specific capture probes include, without limitation, 5xe2x80x2-CCACGCTTGCTTGCTTAAAGACCTC-3xe2x80x2  less than SEQ ID NO. 14 greater than .
In another aspect, the invention is directed to a kit for co-detecting HCV RNA and HIV RNA in a biological sample. The kit comprises:
(a) a pair of oligonucleotide primers specific for the 5xe2x80x2 noncoding region of HCV comprising:
(b) oligonucleotide primers specific for HIV-1 which comprise a forward primer with the sequence:
5xe2x80x2-CTGCTTAAGCCTCAATAAAGCTTGCCTTGA-3xe2x80x2 (JBLTR4)  less than SEQ ID NO. 3 greater than , and a reverse primer specific for HIV-1 selected from the group consisting of
(1) 5xe2x80x2-GGGTCTGAGGGATCTCTAGTTACC AGAGT-3xe2x80x2 (JBLTR6)  less than SEQ ID NO. 4 greater than , and
(2) 5xe2x80x2-TGTTCGGGCGCCACTGCTAGAGA-3xe2x80x2 (JBLTR8)  less than SEQ ID NO. 5 greater than , and (c) oligonucleotide primers specific for HIV-2 which comprise a forward primer with the sequence 5xe2x80x2-GGGAGGTTCTCTCCAGCACTAGCA-3xe2x80x2 (2LTRe)  less than SEQ ID NO. 6 greater than , and a reverse primer specific for HIV-2 with the sequence:
5xe2x80x2-GCGACTAGGAGAGATGGGAACACACA-3xe2x80x2 (2LTR-R1)  less than SEQ ID NO. 7 greater than .
In yet another aspect, the invention is directed to a kit for co-amplifying HCV DNA and HIV DNA in a DNA sample. The kit comprises:
(a) a pair of oligonucleotide primers specific for the 5xe2x80x2 noncoding region of HCV comprising:
(b) oligonucleotide primers specific for HIV-1 which comprise a forward primer with the sequence:
5xe2x80x2-CTGCTTAAGCCTCAATAAAGCTTGCCTTGA-3xe2x80x2 (JBLTR4)  less than SEQ ID NO. 3 greater than , and a reverse primer specific for HIV-1 selected from the group consisting of:
(1) 5xe2x80x2-GGGTCTGAGGGATCTCTAGTTACC AGAGT-3xe2x80x2 (JBLTR6)  less than SEQ ID NO. 4 greater than ,
(2) 5xe2x80x2-TGTTCGGGCGCCACTGCTAGAGA-3xe2x80x2 (JBLTR8)  less than SEQ ID NO. 5 greater than ,and (c) oligonucleotide primers specific for HIV-2 which comprise a forward primer with the sequence 5xe2x80x2-GGGAGGTTCTCTCCAGCACTAGCA-3xe2x80x2 (2LTRe)  less than SEQ ID NO. 6 greater than , and a reverse primer specific for HIV-2 with the sequence:
5xe2x80x2-GCGACTAGGAGAGATGGGAACACACA-3xe2x80x2 (2LTR-R1)  less than SEQ ID NO. 7 greater than .
The present inventors have discovered that simultaneous detection of low levels of Hepatitis C Virus (HCV) RNA and Human Immunodeficiency Virus (HIV) RNA in biological samples containing HCV and HIV RNA can be achieved in a multiplex assay by (i) performing a reverse transcription reaction on RNA derived from the sample and (ii) amplifying the reverse transcription products using particular pairs of oligonucleotides having sequences complementary to certain sequences present in HCV and HIV RNA. The present invention thus provides improved single-round, multiplex reverse transcription/amplification assays which detect low copy levels of HCV and HIV RNA in the same samples.
Oligonucleotide primers are selected based on considerations of sequence conservation, intra- and inter-molecular interactions, and the predicted secondary structures of the amplicon and surrounding sequence. Furthermore, the primers and assay system are designed to allow the co-amplification (and co-detection) of multiple regions of the HCV and HIV genomes, multiple viral species, and an internal positive control (IPC) RNA (or DNA). Simultaneous amplification/detection of multiple regions of the viral genomes increases assay sensitivity and the co-amplification of an IPC decreases the likelihood of false negative results because of PCR inhibition.
Many techniques in molecular biology, microbiology, recombinant DNA, and protein biochemistry are used in practicing the present invention, such as those explained in, for example, Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.. Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed.); Transcription and Translation, 1984 (Hames and Higgins eds.); A Practical Guide to Molecular Cloning, the series, Methods in Enzymology (Academic Press, Inc.); and Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.).
xe2x80x9cNucleic acidxe2x80x9d or xe2x80x9cpolynucleotidexe2x80x9d as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, such as, for example, DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as xe2x80x9cprotein nucleic acidsxe2x80x9d (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
A xe2x80x9ccomplementxe2x80x9d of a nucleic acid sequence as used herein refers to the antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
A xe2x80x9cprimerxe2x80x9d as used herein is an isolated oligonucleotide between about 5 and about 50 nucleotides in length, preferably between about 6 and about 25 nucleotides in length and most preferably between about 6 and about 18 nucleotides in length, that forms a duplex with a single-stranded nucleic acid sequence of interest and allows polymerization of a complementary strand using, e.g., reverse transcriptase or DNA polymerase.
An xe2x80x9cisolatedxe2x80x9d nucleic acid as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring or a reaction mixture if it is synthetic). An isolated nucleic acid typically contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the components with which it was originally associated.
A nucleic acid sequence that is xe2x80x9cderived fromxe2x80x9d a designated sequence refers to a sequence that corresponds to a region of the designated sequence. This encompasses sequences that are homologous or complementary to the sequence.
An internal positive control (IPC) target nucleic acid refers to a synthetic nucleic acid sequence cloned into a plasmid vector which is subsequently linearized, typically by the action of a restriction endonuclease. An IPC will typically have multiple primer binding sequences surrounding a generic probe-binding region, and acts as a generic control against false negative results in nucleic acid amplification reactions.
The sequence of a preferred internal positive control target DNA is:
Nucleic acids comprising any of the sequences disclosed herein or subsequences thereof can be prepared by conventional methods. For example, DNA can be chemically synthesized using, e.g., the phosphoramidite solid support method of Matteucci et al., 1981, J Am. Chem. Soc. 103:3185, the method of Yoo et al., 1989, J. Biol. Chem. 764:17078, or other well known methods. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, xe2x80x9ccapsxe2x80x9d, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. PNAs are also encompassed by the term xe2x80x9cnucleic acidxe2x80x9d. The nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
Amplification as used herein refers to an iterative process by which a nucleic acid is copied. Suitable methods for amplification include without limitation polymerase chain reaction, ligase chain reaction, strand displacement amplification, and nucleic acid single base amplification, and transcription mediated amplification.
The present invention provides methods for detection of HCV and HIV in biological samples. The methods are carried out by
(i) performing a reverse transcription reaction using as a template RNA contained within or derived from the sample;
(ii) amplifying the reverse-transcription products, if any, using at least one pair of amplification primers having sequences corresponding to sequences within the genome of HCV, preferably in the 5xe2x80x2 noncoding region of HCV, and at least one pair of amplification primers having sequences corresponding to sequences within the genome of HIV, to produce HCV-specific and HIV-specific amplification products; and
(iii) detecting the HCV-specific and HIV-specific amplification products. Detection of HCV-specific or HIV-specific amplification products indicates the presence of HCV and HIV RNA, respectively, in the sample.
According to the invention, a biological sample is obtained from a patient by any conventional means. Suitable biological samples include, without limitation, blood, serum, plasma, urine, breast milk, and cerebrospinal fluid. Preferably, plasma is used as the source of viral RNA.
The biological sample is treated in any manner that provides access of the reverse transcription reagents to RNA, specifically viral RNA, contained within the sample. RNA xe2x80x9cderived fromxe2x80x9d a biological sample is any RNA which was originally present in the sample and to which access has been gained by treating the sample. Preferably, RNA is extracted from the sample using any method well known in the art, such as, e.g., methods employing guanidinium thiocyanate, or using commercially available reagents and methods such as, e.g., PureScript∂ from Gentra Systems, Inc. (Minneapolis Minn.). Any extraction procedure may be used that results in separation from the RNA of RNases, other proteins, and/or any other components that might interfere with reverse transcription.
The sample is then subjected to reverse transcription using (a) random primers, such as random hexamer primers obtained from Pharmacia Biotech, Piscataway, N.J., and/or (b) primers derived from the 5xe2x80x2 or 3xe2x80x2 non-coding regions of the HCV RNA genomic sequence. Reverse transcription is carried out using conventional procedures, such as are described in Current Protocols in Molecular Biology, Volumes 1, 11, and 111, 1997 (F. M. Ausubel ed.); in U.S. Pat. No. 5,322,770; in Young, et al., J. Clin. Microbiol. 31(4):882 (1993); Myers et al., Biochemistry 30(3):7661 (1991); or as described in provisional patent application Ser. No. 60/118,520, filed Feb. 3, 1999.
Following the reverse transcription reaction, the products are amplified. Any method for amplification may be used, including, without limitation, polymerase chain reaction (PCR), ligase chain reaction, strand displacement reaction, transcription mediated amplification, or nucleic acid single base amplification. Preferably, PCR is used. Typically, a reaction mixture containing all of the necessary components for PCR is added directly to the reverse transcription reaction mixture. Amplification is then carried out using conditions specified by the primer pairs that are used.
The present inventors have discovered certain pairs of HCV-specific and HIV-specific amplification primers may be used simultaneously to detect low levels of both HCV and HIV RNA in patient samples. Non-limiting examples of primers that may be used in practicing the invention include those listed in Table 1 below.
Following amplification, the amplified products may be detected using any method known in the art, including, without limitation, gel electrophoresis in agarose or acrylamide gels; non-isotopic calorimetric detection such as the SureCell(copyright) system, available from Ortho Clinical Diagnostics, Rochester, N.Y. (see, e.g., Example 1 below); ECi detection; chemiluminescence, and fluorescence.
The detection of viral-specific amplification products indicates the presence of viral RNA in the sample. When gel electrophoresis is used, viral-specific amplification products are confirmed by their size, as predicted by the location in the respective viral RNA of the sequences corresponding to the amplification primers used in the reaction.
The present invention finds use in the diagnosis of HIV and HCV infection in patients; in testing the efficacy of anti-viral therapeutic regimens; and in screening the blood supply for HCV-infected and HIV-infected samples.
The following examples illustrate the present invention without limitation.